The ability to understand and manipulate the biochemical events controlling how cells make decisions is central to the development of novel treatments for microbial infections, autoimmune diseases, cancer, and developmental defects. The bacterium Bacillus subtilis differentiates into stress-resistant, metabolically inert spores upon starvation, and activates a separate gene general stress response pathway when challenged with various stresses. B. subtilis sporulation and stress response are ideal model pathways for which to develop innovative new technologies to study and control differentiation. Though the basic regulatory interactions in the core gene circuits are known, we lack a systems-level understanding of how dynamical changes in the major regulatory proteins result in cellular information processing and the ultimate guide these large-scale cellular decisions. Our central hypothesis is that the ability to dynamically perturb and observe protein activities in gene circuis in real time will yield crucial insights about cellular decision-making and differentiation. To thi end, we propose to develop a technology for interrogating the signaling properties of the B. subtilis sporulation and stress response gene circuits that uses time-varying light signals to program exceptionally well-defined gene expression dynamics in live cells. As a first aim, we will re-engineer a green/red light-switchable two-component system we previously built in E. coli to control transcription and generate dynamical gene expression functions in B. subtilis. By moving this system from E. coli to the evolutionary distant B. subtilis, we will also gain insights on the considerations needed to move these powerful optogenetic tools to other model organisms and clinically important species. As a second aim, we will use our optical method to analyze how different rates of activation of the major sporulation regulators impact the circuit functions and resulting phenotype. We expect to unequivocally demonstrate that i) successful execution of the sporulation program requires not only proper steady-state levels, but also proper dynamics of key regulators and that ii) recently observed pulsing in the major stress response regulator are used to elicit proportional activation levels of many target genes. In the third aim, we will use or optical method to investigate the biological significance of the recently described pulsatile dynamics of the master sporulation regulator at the population and single cell levels. In particular, we will evaluate our recent hypothesis that the sporulation pulses must occur after DNA replication to ensure that spores inherit chromosomes and a prevailing hypothesis that a supra-threshold concentration of the master sporulation regulator must be reached for cells to commit to sporulation. Since the B. subtilis sporulation and stress response circuits are widely conserved among medically important spore-forming bacteria including B. cereus, B. anthracis, and C. difficile, our result will not only enable breakthroughs in the understanding of cellular decision-making, but also provide a basis for design of new antibacterial agents.